Select suppressor mutations increase C5A49 protein abundance. (A) Relative abundance of either the wild-type or mutant C5 protein and Lon protease during early-to-mid exponential phase growth at the permissive (30°C) or sublethal temperature (40°C), as compared to the original A49 parental strain (DA61546, set to 1). Strains DA61546, DA65195, and DA65198 carry the rnpA49 mutant allele (encoding C5A49). (B) Relative abundances of the wild-type C5 protein and Lon protease in lon+ (DA5438) and lon− (DA22599) E. coli MG1655 strains during early-to-mid exponential phase growth at 30°C, as compared to the E. coli MG1655 wild-type control strain (DA5438, set to 1). Strain DA22599 contains a partial deletion of the lon coding region in an E. coli MG1655 genetic background such that the overlapping coding region for the small RNA SraA and the overlapping promoter for the downstream hupB gene remain intact (Nicoloff and Andersson 2013). GadE, a known Lon substrate, is included as a control to demonstrate the loss of Lon activity in DA22599 (Heuveling et al. 2008). For both A and B, total proteome analysis was performed via TMT-based relative quantification using two biological replicates for each strain (at each temperature for A). Briefly, peptides isolated from each sample were labeled with unique isobaric TMT tags, pooled, and analyzed using mass spectrometry. Peptides were identified by matching the resulting ion peaks with those reported in fragment databases and relative peptide abundance was quantified by comparing TMT reporter ion intensities across all samples. Horizontal bars represent the mean between the two biological replicates; vertical I-bars represent the range. Some data points overlap.
